Argonaute (Ago) 蛋白是一種蛋白質家族，存在於原核以及真核生物中，是負責作用於基因調控、外來物質免疫反應的重要蛋白。 在嗜熱細菌中，Ago 蛋白擁有識別並切割去氧核醣核酸分子的能力，這對細菌本身的基因調控、免疫反應和病毒防禦很重要。根據先前的研究，Ago蛋白在醫學方面有廣泛的應用潛力，例如應用於基因治療和檢測。 其中RADAR（Renewed-gDNA Assisted DNA cleavage by Argonaute）是一種新型Ago蛋白所媒介的檢測方法。此Ago蛋白亦有潛力去實現高效、高精度的基因治療。

在本研究中，我們以GBS（B 組鏈球菌）中用於編碼 CAMP 因子的 Cfb基因，做為我們檢測的目標。GBS菌會透過母體傳染給胎兒並造成致命性的新生兒敗血症。檢測方法是透過使用 PfAgo 介導的檢測，我們首先表達 PfAgo並驗證了PfAgo核酸內切酶活性。為了檢測Cfb基因，我們設計了三組gDNA，並測試出其中的G2是功能性最優秀的。此外，我們在實驗過程中亦發現若gDNA引導PfAgo切割結果為Blunt端，切割效率會比切割出粘性端差。以及當靶向不同長度的目標基因時，同一組 gDNA 引導的切割效率會有所不同。

最後，我們使用PfAgo的兩步驟切割，並進行了涉及八個與 GBS 無關的基因組以及來自不同來源的九個不同 GBS 基因組的實驗。並利用電泳或是帶有螢光的基因來顯示檢驗的結果。我們在反應中成功區分了正、負兩個組別，達到提高檢測B 組鏈球菌能力以及提高檢測特異性與準確性的目的。

Argonaute (Ago) proteins are a family of proteins that exist in both prokaryotic and eukaryotic organisms and play crucial roles in gene regulation and immune responses to foreign substances. In thermophilic bacteria, Ago proteins have the ability to recognize and cleave DNA molecules, which is important for bacterial gene regulation, immune responses, and viral defense. Previous studies have indicated the wide potential applications of Ago proteins in the medical field, such as gene therapy and detection. One novel detection method mediated by Ago proteins is RADAR (Renewed-gDNA Assisted DNA cleavage by Argonaute), which has the potential to achieve efficient and precise gene therapy.

In this study, we targeted the Cfb gene encoding the CAMP factor in Group B Streptococcus (GBS) as our detection target. GBS can be transmitted from the mother to the fetus, causing life-threatening neonatal sepsis. The detection method involved the use of PfAgo-mediated detection. We first expressed PfAgo and validated its endonuclease activity. To detect the Cfb gene, we designed three sets of gDNA and found that G2 exhibited the best functional performance. Additionally, during the experiment, we observed that if gDNA guides PfAgo to cleave resulting in blunt ends, the cutting efficiency is lower compared to guiding the cleavage to produce sticky ends. Moreover, the cutting efficiency of the same set of gDNA guides varied when targeting different lengths of target genes.

Last but not least, we conducted experiments involving eight genomes unrelated to GBS and nine different GBS genomes from different sources using PfAgo two-step cleavage. The results were displayed through gel electrophoresis or fluorescently labeled genes. We successfully distinguished the positive and negative groups in the reaction, achieving the goal of improving the detection capability, specificity, and accuracy for GBS detection.

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